Organization of the Cytoskeleton in Ectopic Foci of the Endometrium with Rare Localization

(1) Background: Endometriosis is a common pathology of the female reproductive system, often accompanied by pain and decreased fertility. However, its pathogenesis has not been sufficiently studied regarding the role of the cytoskeleton. In this study, we describe two clinical cases involving rare localization of extragenital endometriosis (umbilicus) and compare them with genital endometriosis of different localization (ovaries and uterus), as well as eutopic endometrium obtained with separate diagnostic curettage without confirmed pathology. (2) Methods: The relative content of actin and tubulin cytoskeleton proteins was determined by Western blotting, and the expression of genes encoding these proteins was determined by RT-PCR in the obtained intraoperative biopsies. The content of 5hmC was estimated by dot blot experiments, and the methylase/demethylase and acetylase/deacetylase contents were determined. (3) Results: The obtained results indicate that the content of the actin-binding protein alpha-actinin1 significantly increased (p < 0.05) in the groups with endometriosis, and this increase was most pronounced in patients with umbilical endometriosis. In addition, both the mRNA content of the ACTN1 gene and 5hmC content increased. It can be assumed that the increase in 5hmC is associated with a decrease in the TET3 demethylase content. Moreover, in the groups with extragenital endometriosis, alpha- and beta-tubulin content was decreased (p < 0.05) compared to the control levels. (4) Conclusions: In analyzing the results, further distance of ectopic endometrial foci from the eutopic localization may be associated with an increase in the content of alpha-actinin1, probably due to an increase in the expression of its gene and an increase in migration potential. In this case, a favorable prognosis can be explained by a decrease in tubulin content and, consequently, a decrease in the rate of cell division.

Analysis of Water-Soluble Proteins by Two-Dimensional Electrophoresis in the Encystment Process of Colpoda cucullus Nag-1 and Cytoskeletal Dynamics

Assays of protein contained in water-soluble fraction of encysting cells Colpoda cucullus Nag-1 by two-dimensional electrophoresis (2-D PAGE) and mass spectrometry (MS) revealed that the amount of β-tubulin abruptly increased in 2.5–10 h after encystment induction. Judging from the results that total α-tubulin content did not decrease much until 12 h after encystment induction, the result indicates that disassembly of microtubules may occur soon after encystment is induced. Therefore, we tried to visualize dynamics of microtubules. Immunofluorescence microscopy using anti-α-tubulin antibody indicated that disassembly of axonemal microtubules of cilia became within 1.5 h after encystment induction, and resorbed in 3 days. Although the cytoplasmic microtubules failed to be visualized clearly, encystmentdependent globulation of cells was promoted by taxol, an inhibitor of disassembly of microtubules. It is possible that a temporary formation of cytoplasmic microtubules may be involved in cell globulation. The phosphorylation level of actin (43 kDa) became slightly elevated just after encystment induction. Lepidosomes, the sticky small globes surrounding encysting cells, were vividly stained with Acti-stain 555 phalloidin, suggesting that 43-kDa actin or its homologues may be contained in lepidosomes.

Cytoskeleton Structure in Mouse Sperm and Testes After 30 Days of Hindlimb Unloading and 12 Hours of Recovery

Background/Aims: Changes in the external mechanical field result in cytoskeleton reorganization and the formation of adaptive patterns in different types of cells, including somatic cells and sex cells. The aim of this research was to study the protein and mRNA content of cytoskeletal and sperm-specific genes in the sperm and testis cells of mice. Methods: Mice were subjected to 30 days of antiorthostatic suspension to simulate weightlessness, followed by 12 h of recovery, while receiving essential phospholipids at a dosage of 500 mg/kg/day (30HSE and 30HSE+12h groups) or a similar dosage of a placebo (30HS and 30HS+12h groups). Accordingly, reference groups (CE group and C group) were formed. The total number and the percentage of motile spermatozoa were calculated using a Makler chamber. To analyze the number of viable spermatozoa and the permeability of their membranes, eosin staining was used as well as Diff-Quick for a morphological evaluation. Relative protein and mRNA content was estimated in a western blot and quantitative PCR assay, respectively. Results: The relative protein expression levels of actin (beta and gamma) and two alpha-actinin isoforms (1 and 4) remained constant in the sperm of all study groups, except for the 30HS+12h group, where the alpha-actinin-4 level was 13% higher than in the reference group (p < 0.1). In the testis cells, the relative actin isoform content was equivalent to that in the spermatozoa. However, in the testis cells, the ACTN1 mRNA content was 17% higher in the 30HS group than in the C group (p < 0.05), and decreased after 12 h of recovery. In contrast, the ACTN4 mRNA content was 20% lower in the 30HS group than in the reference group (p < 0.05) and increased after the 12-h recovery period. At the same time, in the group administered the essential phospholipids, the relative ACTN1 and ACTN4 mRNA content did not differ from those of the reference group. The relative beta-tubulin content was similar in the reference C group and the reference CE group, which was administered the essential phospholipids. In the 30HS and 30HS+12h groups, the beta-tubulin content decreased by 19% and 22% (p < 0.05), respectively, and they also decreased in the groups administered the essential phospholipids (30HSE and 30HSE+12h groups, by 27% and 33%, respectively, p < 0.05). In the testis tissue, the relative tubulin content did not change in any of the experimental groups. At the same time, the relative mRNA content of the genes encoding the studied cytoskeletal proteins increased, which may indicate the protein content was regulated mainly at the translational level. Conclusion: The spermogram parameters and the content of the sperm-specific proteins and the associated mRNAs revealed a decrease in the number of mature spermatozoa in mice suspended under conditions of weightlessness. Moreover, the decrease was prevented by the administration of essential phospholipids.

Triiodothyronine Rapidly Alters the TSH Content and the Secretory Granules Distribution in Male Rat Thyrotrophs by a Cytoskeleton Rearrangement-Independent Mechanism

Rapid actions of T3 on TSH synthesis in posttranscriptional steps, such as polyadenylation and translation rate, have already been described. The focus of this paper was to characterize rapid actions of T3 on TSH secretion and the involvement of actin and microtubule cytoskeleton in this process. For that, sham-operated (SO) and thyroidectomized (Tx) rats were subjected to acute or chronic treatment with T3. We observed a disarrangement in microtubule and actin cytoskeletons and an increase in Tshb mRNA levels in Tx rats, whereas the total TSH protein content was reduced in the pituitary gland as a whole, but increased in the secretory granules close to the plasma membrane of thyrotrophs, as well as in the extracellular space. The acute T3 dose promoted a rapid increase and redistribution of TSH secretory granules throughout the cytoplasm, as well as a rearrangement in actin and microtubule cytoskeletons. The T3 chronic treatment outcome reinforces the acute effects observed and, additionally, evinces an increase in the α-tubulin content and a rearrangement in microtubule cytoskeleton. Thus, T3 is able to rapidly suppress TSH secretion and, in parallel, to promote a rearrangement in actin and microtubules assembly throughout the pituitary gland, effects that seem to be independent from each other.

Paclitaxel-dependent mutants have severely reduced microtubule assembly and reduced tubulin synthesis

A subset of mutant cell lines selected for resistance to the antitumor drug paclitaxel are unable to progress normally through mitosis unless the drug is present in the growth medium. Without paclitaxel the cells form defective spindles, undergo aberrant mitoses, fail to complete cell division and eventually die. Analysis of these drug-dependent cells revealed a low amount of microtubule polymer and less tubulin production than wild-type cells. Ribonuclease protection experiments indicated that the decreased tubulin protein was due to decreased tubulin mRNA. Enhancing microtubule assembly by treating the cells with paclitaxel, restored tubulin to levels comparable with those of paclitaxel-treated wild-type cells, which demonstrated that the drug-dependent cells do not have a permanent impairment in their capacity to synthesize tubulin. Paclitaxel-resistant (but not dependent) cells have a smaller reduction in microtubule polymer with little or no decrease in tubulin production, whereas colcemidresistant cells have increased microtubule assembly but also exhibit little or no change in tubulin production. Finally,a mutant cell line producing an unstable β-tubulin protein has normal growth as well as normal synthesis and polymerization of tubulin, despite an approximately 30% decrease in steady state tubulin content. These studies establish a lower limit of tubulin assembly needed for cell survival and indicate that tubulin assembly must fall below this point to trigger a significant decrease in tubulin synthesis.